Our long-term goal is a deeper understanding of how bacterial fitness and virulence intersect in pathogenesis. The present proposal focuses on the role of the Staphylococcal virulon in infections caused by community- acquired, methicillin-resistant Staphylococcus aureus (CA-MRSA). While the outcome of a CA-MRSA encounter is usually asymptomatic colonization, the propensity of CA-MRSA strains to produce invasive infection defines a capacity to resist host innate immune clearance mechanisms. The CA-MRSA factors responsible for the diverse outcomes of this important host-pathogen interaction are unknown. The agr locus, a global regulator of virulence gene expression, has received much attention due to its critical role in invasiveness in animal models. Our preliminary data suggesting that invasion of CA-MRSA in humans is accompanied by enrichment for variants with enhanced agr activity supports this idea and indicates that the early stages of disease are accompanied by bacterial changes that give rise to an invasive-disease phenotype. DNA sequencing of such variants revealed that the agr locus was unaltered, indicating that upstream agr regulation contributes to the phenotype. Dysfunction of the alternative sigma factor sigB was identified as a frequent cause of the phenotype, but our preliminary survey of clinical isolates indicates the occurrence of alterations in other, unknown genes. Critically, variants had a fitness advantage in vitro, suggesting that selective pressures independent of host factors can account in part for their emergence. These considerations give rise to the following two-part hypothesis, upon which the present proposal is based: 1) through mutation of genes, S. aureus frequently generates within an infection a group of clones differing in their virulence, and 2) the shift of CA-MRSA strains from a commensal to an invasive-disease phenotype is associated with selection in vivo for mutations that enhance virulence. To test the hypothesis, we will 1) determine the frequency of within-host upshifts in agr function during clinical CA-MRSA skin infection and invasive disease, 2) characterize the genetic basis of these upshifts, and 3) evaluate the relative virulence and fitness of strains with upshifts both in vitro and in vivo using serial passage and competition tests. Field studies will focus on community subjects, where the barrier to infection is higher than in hospitalized patients for whom disruption of barrier functions by disease and clinical intervention permit S. aureus strains that lack full virulence to cause infection. The proposed work will impact critical questions in infectious disease research, such as the best use of therapeutic modalities that are being developed to target agr and virulence, as well as more basic yet closely intertwined problems, such as the ways in which S. aureus transitions from an innocent member of our normal flora to an invasive, life-threatening pathogen. The work will have broad implications for forestalling such events.